Cancer is the second leading cause of death in the U.S., and is responsible for losses of over $100 billion per year due to medical costs and lost productivity. Abnormalities in the Epidermal Growth Factor Receptor (EGFR) system are recognized as a major cause of cancers and significant biomedical effort has been directed towards this target in a search for novel anti-cancer agents. In Phase I of this project we proposed the re-engineering of EGF to transform it from an agonist to an antagonist as the first stage of our Anti Cancer Ligand program. Computational analysis of the EGF sequence and scrutiny of EGF/EGFR interactions led to a rational program of site-directed mutagenesis, production, purification and analysis of EGF variants culminating in the successful accomplishment of the goals established in our Phase I SBIR grant, the identification of our early stage lead, a single point mutant with antagonist properties. In Phase II of this project we will confirm the therapeutic potential of that lead in mouse xenograft studies using the high-EGFR human cancer A431 cell line and then improve upon its potential through a program of affinity maturation using both phage display and rational modification. The high affinity antagonist will be tested in both in vitro and in vivo assays to demonstrate its efficacy against human tumor models and to demonstrate that its mode of action is consistent with its therapeutic properties. The identification of this pre-clinical drug lead is the ultimate goal of this proposed Phase II program, and the intellectual property generated in the course of this program will provide the basis for marketing this lead to the pharmaceutical industry.